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CN111690587B - 一种离心筛选具有高含油率油脂酵母菌株的方法及其应用 - Google Patents

一种离心筛选具有高含油率油脂酵母菌株的方法及其应用 Download PDF

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CN111690587B
CN111690587B CN201910186710.0A CN201910186710A CN111690587B CN 111690587 B CN111690587 B CN 111690587B CN 201910186710 A CN201910186710 A CN 201910186710A CN 111690587 B CN111690587 B CN 111690587B
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鲍杰
赵瞭
侯伟亮
胡明珊
张建
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Shanxi Institute Of Synthetic Biology Co ltd
Cathay R&D Center Co Ltd
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Abstract

本发明公开了一种离心筛选具有高含油率油脂酵母菌株的方法及其应用，属于生物技术和微生物技术领域。具体步骤为将油脂酵母菌株培养在木质纤维素水解液中，通过离心发酵液获取上层包含高油脂含量的轻油脂细胞转接后继续培养，并逐步提高离心力直到菌株发酵性能达到稳定。筛选得到新菌株命名为皮状丝孢酵母MS28,其保藏编号为CGMCC No.14780。本发明快速高效，对所有油脂酵母菌株筛选均具有重要借鉴意义。

Description

一种离心筛选具有高含油率油脂酵母菌株的方法及其应用
【技术领域】
本发明涉及生物技术和微生物技术领域，一种离心筛选具有高含油率油脂酵母菌株的方法及其应用。
【背景技术】
油脂是生产高能量密度生物燃料比如生物柴油或者航空燃料的重要前提物质。来自于大豆油菜籽，向日葵或者棕榈的植物油是进行生物柴油和航空燃料生产的主要来源。然而，极大的生物柴油市场需求需要稳定快速的底物供应。可再生的木质纤维素是非常适宜的原料来源进行发酵法生产生物柴油。
发酵菌株是影响油脂生产的关键因素，其应该具有高木质纤维素来源抑制物的耐受性，多种碳源利用行为和高油脂富集能力。因此，菌株选育尤为重要。油脂酵母菌株选育方法主要包括浅蓝菌素染色筛选，直接油脂测重筛选，微型生物反应器筛选和漂浮细胞筛选。而人工智能辅助的高通量筛选尽管增加了筛选效率但仍远低于细胞数，通常仅有1％的细胞得到筛选，而且后续的称重油脂测定极大延迟了整个筛选速率。
本发明提出了一种基于离心过程中细胞密度差异性分布的原理简单快速筛选含油率高的轻油脂细胞菌株。此方法的应用不仅可以简化常规筛选的繁琐过程也可以极大提升高产油脂细胞的筛选效率。
【发明内容】
本发明的目的在于提供一种离心筛选具有高含油率油脂酵母菌株的方法。
本发明的目的是通过以下技术方案来实现的：
一种离心筛选具有高含油率油脂酵母菌株的方法及其应用，其具体步骤为：
(1)将亲本菌株以10％接种量接种于50ml 15％玉米秸秆水解液中，在30℃和180rpm条件下培养72h，其添加营养盐组分包括1.0 g/L KH2PO4,0.5g/L酵母提取物,0.5g/LMgSO4 7H2O,0.22g/L (NH4)2SO4；
(2)取25ml发酵液在一定离心力下离心2min，并吸取5ml 发酵液最上层清液转至新鲜水解液中在相同条件下进行培养；
(3)随着转接的进行逐渐增加离心力，直到菌株发酵性能达到稳定。
本发明人利用离心条件下细胞密度分布差异的原理实现了具有高含油率的轻细胞筛选。这种简单高效方法的应用不仅极大提高了油脂酵母菌株筛选的效率也降低筛选操作的难度。
【附图说明】
图1离心筛选过程曲线；
图2亲本菌株和筛选菌株在15％水解液中摇瓶发酵比较；
图3亲本菌株和筛选菌株在20％水解液中3L罐发酵比较；
图4亲本菌株和筛选菌株在30％固含量同步糖化共发酵中5L 罐发酵比较。
【具体实施方式】
以下提供本发明一种离心筛选具有高含油率油脂酵母菌株的方法及其应用的具体实施方式。
实施例1菌株筛选
首先将皮状丝孢酵母亲本菌株接种到15％玉米秸秆水解液中于 30℃和180rpm培养72h，其中添加营养盐组分包括1.0g/L KH2PO4, 0.5g/L酵母提取物,0.5g/L MgSO4 7H2O,0.22g/L(NH4)2SO4；而后对发酵液进行离心并取离心上清液以10％接种量接种于新的水解液中在相同条件下进行培养；并逐步提高离心力从1000×g到12000×g 筛选最轻油脂细胞。最终筛选得到菌株命名为皮状丝孢酵母(Trichosporoncutaneum)MS28 并已于2017年10月11日保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏编号为CGMCC No.14780。
实施例2 15％水解液摇瓶发酵
筛选菌株发酵性能首先在摇瓶规模进行测试。发酵菌株以10％接种量接种于包含50ml 15％水解液的摇瓶中在30℃,180rpm条件下培养72h,其添加营养盐组分包括1.0g/LKH2PO4,0.5g/L酵母提取物,0.5g/L MgSO4 7H2O,0.22g/L(NH4)2SO4。发酵结果表明筛选菌株发酵性能获得了显著提升，最终获得含油率和油脂浓度相比于亲本菌株分别提高了3.2倍和2.5倍。
实施例3 20％水解液3L发酵
发酵菌株以10％接种量接种于包含1L 20％水解液的3L发酵罐中在30℃,600rpm和1vvm通气条件下培养72h；发酵过程中pH 维持在5.0，发酵中添加营养盐组分包括1.0g/LKH2PO4,0.5g/L酵母提取物,0.5g/L MgSO4 7H2O,0.22g/L(NH4)2SO4。发酵结果表明相比于亲本菌株，筛选菌株的含油率从17.1％增加到了44.5％，油脂浓度从3.3g/L增加到了15.5g/L。
实施例4 30％固含量同步糖化与发酵
预处理和脱毒后物料首先经过12h预糖化而后以10％接种量接入发酵菌株。同步糖化与发酵在30℃,600rpm和1vvm通气条件下持续96h；发酵过程中pH维持在5.0，发酵中添加营养盐组分包括 1.0g/L KH2PO4,0.5g/L酵母提取物,0.5g/L MgSO4 7H2O,0.22g/L(NH4)2SO4。发酵结果表明相比于亲本菌株，筛选菌株具有更强的碳源利用能力，其可以完全利用木质纤维素来源的全部糖类，而亲本菌株仅可以部分利用葡萄糖。最终筛选菌株获得了34.4g/L的油脂累积而亲本菌株只获得了7.5g/L油脂产量。

Claims (1)

1.一株皮状丝孢酵母（Trichosporoncutaneum）MS28，其特征在于，其保藏编号为CGMCCNo.14780。
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